Reduced C9orf72 function leads to defective synaptic vesicle release and neuromuscular dysfunction in zebrafish.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) is a hexanucleotide repeat expansion within the C9orf72 gene. Reduced levels of C9orf72 mRNA and protein have been found in ALS/FTD patients, but the role of this protein in disease pathogenesis is still poorly understood. Here, we report the generation and characterization of a stable C9orf72 loss-of-function (LOF) model in the zebrafish. We show that reduced C9orf72 function leads to motor defects, muscle atrophy, motor neuron loss and mortality in early larval and adult stages. Analysis of the structure and function of the neuromuscular junctions (NMJs) of the larvae, reveal a marked reduction in the number of presynaptic and postsynaptic structures and an impaired release of quantal synaptic vesicles at the NMJ. Strikingly, we demonstrate a downregulation of SV2a upon C9orf72-LOF and a reduced rate of synaptic vesicle cycling. Furthermore, we show a reduced number and size of Rab3a-postive synaptic puncta at NMJs. Altogether, these results reveal a key function for C9orf72 in the control of presynaptic vesicle trafficking and release at the zebrafish larval NMJ. Our study demonstrates an important role for C9orf72 in ALS/FTD pathogenesis, where it regulates synaptic vesicle release and neuromuscular functions.

TMEM106B modifies TDP-43 pathology in human ALS brain and cell-based models of TDP-43 proteinopathy.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43) inclusions (FTLD-TDP) share the neuropathological hallmark of aggregates of TDP-43. However, factors governing the severity and regional distribution of TDP-43 pathology, which may account for the divergent clinical presentations of ALS and FTLD-TDP, are not well understood. Here, we investigated the influence of genotypes at TMEM106B, a locus associated with risk for FTLD-TDP, and hexanucleotide repeat expansions in C9orf72, a known genetic cause for both ALS and FTLD-TDP, on global TDP-43 pathology and regional distribution of TDP-43 pathology in 899 postmortem cases from a spectrum of neurodegenerative diseases. We found that, among the 110 ALS cases, minor (C)-allele homozygotes at the TMEM106B locus sentinel SNP rs1990622 had more TDP-43 pathology globally, as well as in select brain regions. C9orf72 expansions similarly associated with greater TDP-43 pathology in ALS. However, adjusting for C9orf72 expansion status did not affect the relationship between TMEM106B genotype and TDP-43 pathology. To elucidate the direction of causality for this association, we directly manipulated TMEM106B levels in an inducible cell system that expresses mislocalized TDP-43 protein. We found that partial knockdown of TMEM106B, to levels similar to what would be expected in rs1990622 C allele carriers, led to development of more TDP-43 cytoplasmic aggregates, which were more insoluble, in this system. Taken together, our results support a causal role for TMEM106B in modifying the development of TDP-43 proteinopathy.

C9orf72 ALS-FTD: recent evidence for dysregulation of the autophagy-lysosome pathway at multiple levels.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two clinically distinct classes of neurodegenerative disorders. Yet, they share a range of genetic, cellular, and molecular features. Hexanucleotide repeat expansions (HREs) in the C9orf72 gene and the accumulation of toxic protein aggregates in the nervous systems of the affected individuals are among such common features. Though the mechanisms by which HREs cause toxicity is not clear, the toxic gain of function due to transcribed HRE RNA or dipeptide repeat proteins (DPRs) produced by repeat-associated non-AUG translation together with a reduction in C9orf72 expression are proposed as the contributing factors for disease pathogenesis in ALS and FTD. In addition, several recent studies point toward alterations in protein homeostasis as one of the root causes of the disease pathogenesis. In this review, we discuss the effects of the C9orf72 HRE in the autophagy-lysosome pathway based on various recent findings. We suggest that dysfunction of the autophagy-lysosome pathway synergizes with toxicity from C9orf72 repeat RNA and DPRs to drive disease pathogenesis.Abbreviation: ALP: autophagy-lysosome pathway; ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ASO: antisense oligonucleotide; C9orf72: C9orf72-SMCR8 complex subunit; DENN: differentially expressed in normal and neoplastic cells; DPR: dipeptide repeat protein; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; ER: endoplasmic reticulum; FTD: frontotemporal dementia; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HRE: hexanucleotide repeat expansion; iPSC: induced pluripotent stem cell; ISR: integrated stress response; M6PR: mannose-6-phosphate receptor, cation dependent; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: neurodegenerative disorder; RAN: repeat-associated non-ATG; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SLC66A1/PQLC2: solute carrier family 66 member 1; SMCR8: SMCR8-C9orf72 complex subunit; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; WDR41: WD repeat domain 41.

Absence of Survival and Motor Deficits in 500 Repeat C9ORF72 BAC Mice.

A hexanucleotide repeat expansion at C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Initial studies of bacterial artificial chromosome (BAC) transgenic mice harboring this expansion described an absence of motor and survival phenotypes. However, a recent study by Liu and colleagues described transgenic mice harboring a large repeat expansion (C9-500) and reported decreased survival and progressive motor phenotypes. To determine the utility of the C9-500 animals for understanding degenerative mechanisms, we validated and established two independent colonies of transgene carriers. However, extended studies of these animals for up to 1 year revealed no reproducible abnormalities in survival, motor function, or neurodegeneration. Here, we propose several potential explanations for the disparate nature of our findings from those of Liu and colleagues. Resolving the discrepancies we identify will be essential to settle the translational utility of C9-500 mice. This Matters Arising paper is in response to Liu et al. (2016), published in Neuron. See also the response by Nguyen et al. (2020), published in this issue.

Modelling frontotemporal dementia using patient-derived induced pluripotent stem cells.

Frontotemporal dementia (FTD) describes a group of clinically heterogeneous conditions that frequently affect people under the age of 65 (Le Ber et al., 2013). There are multiple genetic causes of FTD, including coding or splice-site mutations in MAPT, GRN mutations that lead to haploinsufficiency of progranulin protein, and a hexanucleotide GGGGCC repeat expansion in C9ORF72. Pathologically, FTD is characterised by abnormal protein accumulations in neurons and glia. These aggregates can be composed of the microtubule-associated protein tau (observed in FTD with MAPT mutations), the DNA/RNA-binding protein TDP-43 (seen in FTD with mutations in GRN or C9ORF72 repeat expansions) or dipeptide proteins generated by repeat associated non-ATG translation of the C9ORF72 repeat expansion. There are currently no disease-modifying therapies for FTD and the availability of in vitro models that recapitulate pathologies in a disease-relevant cell type would accelerate the development of novel therapeutics. It is now possible to generate patient-specific stem cells through the reprogramming of somatic cells from a patient with a genotype/phenotype of interest into induced pluripotent stem cells (iPSCs). iPSCs can subsequently be differentiated into a plethora of cell types including neurons, astrocytes and microglia. Using this approach has allowed researchers to generate in vitro models of genetic FTD in human cell types that are largely inaccessible during life. In this review we explore the recent progress in the use of iPSCs to model FTD, and consider the merits, limitations and future prospects of this approach.

Autophagy in motor neuron diseases.

Motor neuron diseases (MNDs) are a wide group of neurodegenerative disorders characterized by the degeneration of a specific neuronal type located in the central nervous system, the motor neuron (MN). There are two main types of MNs, spinal and cortical MNs and depending on the type of MND, one or both types are affected. Cortical MNs innervate spinal MNs and these control a variety of cellular targets, being skeletal muscle their main one which is also affected in MNDs. A correct functionality of autophagy is necessary for the survival of all cellular types and it is particularly crucial for neurons, given their postmitotic and highly specialized nature. Numerous studies have identified alterations of autophagy activity in multiple MNDs. The scientific community has been particularly prolific in reporting the role that autophagy plays in the most common adult MND, amyotrophic lateral sclerosis, although many studies have started to identify physiological and pathological functions of this catabolic system in other MNDs, such as spinal muscular atrophy and spinal and bulbar muscular atrophy. The degradation of selective cargo by autophagy and how this process is altered upon the presence of MND-causing mutations is currently also a matter of intense investigation, particularly regarding the selective autophagic clearance of mitochondria. Thorough reviews on this field have been recently published. This chapter will cover the current knowledge on the functionality of autophagy and lysosomal homeostasis in the main MNDs and other autophagy-related topics in the MND field that have risen special interest in the research community.

C9ORF72 protein function and immune dysregulation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing disease that affects upper and lower motor neurons eventually leading to paralysis and death by respiratory dysfunction. The most common genetic variant among ALS patients is a hexanucleotide repeat expansion within the first intron of the gene C9ORF72. This expansion elicits a complex cascade of events as a result of both gain- and loss-of-function mechanisms that contribute to neurodegeneration. Increasing evidence suggests that this repeat expansion in C9ORF72 also influences the immune homeostasis. In this review, we consolidate the current understanding of C9ORF72-mediated pathogenesis in both the central nervous system and peripheral immune system and propose mechanisms by which the immune system contributes to ALS.

Disrupted neuronal trafficking in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset neurodegenerative disease caused by degeneration of motor neurons in the brain and spinal cord leading to muscle weakness. Median survival after symptom onset in patients is 3-5 years and no effective therapies are available to treat or cure ALS. Therefore, further insight is needed into the molecular and cellular mechanisms that cause motor neuron degeneration and ALS. Different ALS disease mechanisms have been identified and recent evidence supports a prominent role for defects in intracellular transport. Several different ALS-causing gene mutations (e.g., in FUS, TDP-43, or C9ORF72) have been linked to defects in neuronal trafficking and a picture is emerging on how these defects may trigger disease. This review summarizes and discusses these recent findings. An overview of how endosomal and receptor trafficking are affected in ALS is followed by a description on dysregulated autophagy and ER/Golgi trafficking. Finally, changes in axonal transport and nucleocytoplasmic transport are discussed. Further insight into intracellular trafficking defects in ALS will deepen our understanding of ALS pathogenesis and will provide novel avenues for therapeutic intervention.

The ALS-FTD-linked gene product, C9orf72, regulates neuronal morphogenesis via autophagy.

Mutations in C9orf72 leading to hexanucleotide expansions are the most common genetic causes for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A phenotype resembling ALS and FTD is seen in transgenic mice overexpressing the hexanucleotide expansions, but is absent in C9orf72-deficient mice. Thus, the exact function of C9orf72 in neurons and how loss of C9orf72 may contribute to neuronal dysfunction remains to be clearly defined. Here, we showed that primary hippocampal neurons cultured from c9orf72 knockout mice have reduced dendritic arborization and spine density. Quantitative proteomic analysis identified C9orf72 as a component of the macroautophagy/autophagy initiation complex composed of ULK1-RB1CC1-ATG13-ATG101. The association was mediated through the direct interaction with ATG13 via the isoform-specific carboxyl-terminal DENN and dDENN domain of C9orf72. Furthermore, c9orf72 knockout neurons showed reduced LC3-II puncta accompanied by reduced ULK1 levels, suggesting that loss of C9orf72 impairs basal autophagy. Conversely, wild-type neurons treated with a ULK1 kinase inhibitor showed a dose-dependent reduction of dendritic arborization and spine density. Furthermore, expression of the long isoform of human C9orf72 that interacts with the ULK1 complex, but not the short isoform, rescues autophagy and the dendritic arborization phenotypes of c9orf72 knockout neurons. Taken together, our data suggests that C9orf72 has a cell-autonomous role in neuronal and dendritic morphogenesis through promotion of ULK1-mediated autophagy.

A C9orf72-CARM1 axis regulates lipid metabolism under glucose starvation-induced nutrient stress.

Cells undergo metabolic adaptation during environmental changes by using evolutionarily conserved stress response programs. This metabolic homeostasis is exquisitely regulated, and its imbalance could underlie human pathological conditions. We report here that C9orf72, which is linked to the most common forms of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a key regulator of lipid metabolism under stress. Loss of C9orf72 leads to an overactivation of starvation-induced lipid metabolism that is mediated by dysregulated autophagic digestion of lipids and increased de novo fatty acid synthesis. C9orf72 acts by promoting the lysosomal degradation of coactivator-associated arginine methyltransferase 1 (CARM1), which in turn regulates autophagy-lysosomal functions and lipid metabolism. In ALS/FTD patient-derived neurons or tissues, a reduction in C9orf72 function is associated with dysregulation in the levels of CARM1, fatty acids, and NADPH oxidase NOX2. These results reveal a C9orf72-CARM1 axis in the control of stress-induced lipid metabolism and implicates epigenetic dysregulation in relevant human diseases.

C9orf72 Dipeptide Repeats Cause Selective Neurodegeneration and Cell-Autonomous Excitotoxicity in Drosophila Glutamatergic Neurons.

The arginine-rich dipeptide repeats (DPRs) are highly toxic products from the C9orf72 repeat expansion mutations, which are the most common causes of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the effects of DPRs in the synaptic regulation and excitotoxicity remain elusive, and how they contribute to the development of FTD is primarily unknown. By expressing DPRs with different toxicity strength in various neuronal populations in a Drosophila model, we unexpectedly found that Glycine-Arginine/Proline-Arginine (GR/PR) with 36 repeats could lead to neurodegenerative phenotypes only when they were expressed in glutamatergic neurons, including motor neurons. We detected increased extracellular glutamate and intracellular calcium levels in GR/PR-expressing larval ventral nerve cord and/or adult brain, accompanied by significant increase of synaptic boutons and active zones in larval neuromuscular junctions. Inhibiting the vesicular glutamate transporter expression or blocking the NMDA receptor in presynaptic glutamatergic motor neurons could effectively rescue the motor deficits and shortened life span caused by poly GR/PR, thus indicating a cell-autonomous excitotoxicity mechanism. Therefore, our results have revealed a novel mode of synaptic regulation by arginine-rich C9 DPRs expressed at more physiologically relevant toxicity levels and provided a mechanism that could contribute to the development of C9-related ALS and FTD.SIGNIFICANCE STATEMENT C9orf72 dipeptide repeats (DPRs) are key toxic species causing ALS/FTD, but their roles in synaptic regulation and excitotoxicity are unclear. Using C9orf72 DPRs with various toxicity strength, we have found that the arginine-rich DPRs cause selective degeneration in Drosophila glutamatergic neurons and revealed an NMDA receptor-dependent cell-autonomous excitotoxicity mechanism. Therefore, this study has advanced our understanding of C9orf72 DPR functions in synaptic regulation and excitotoxicity and provided a new mechanism that could contribute to the development of C9-related ALS and FTD.

The epidemiology and genetics of Amyotrophic lateral sclerosis in China.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder associated with loss of motor neurons. Previous knowledge of the disease has been mainly based on studies from Caucasian ALS patients of European descent. Here we review the epidemiological characteristics of ALS among the Chinese population in order to compare the similarities and differences between Chinese ALS cases and those from other countries. We describe a potential lower incidence and prevalence of ALS, a younger age of onset and a lower proportion of familial ALS cases in the Chinese population. Additionally, we highlight potential genetic differences between Chinese and Caucasian ALS patients. Most notably, the frequency of GGGGCC repeat expansions in C9ORF72 in Chinese ALS is significantly lower than in Caucasians. Since some conclusions might not be consistent across all of the studies around China to date, we suggest that it is necessary to carry out a prospective population-based study and large-scale gene sequencing around to better define epidemiological and genetic features of Chinese ALS patients.